(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoic acid monoargininyl salt

ABSTRACT

This invention relates to a new monoargininyl salt form of the endothelin receptor antagonist (E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoic acid.

This application is a 371 of PCT/US98/25903, filed on Dec. 7, 1998,which claims benefit of Provisional Application Ser. No. 60/067,732filed Dec. 8, 1997.

FIELD OF THE INVENTION

This invention relates to a new monoargininyl salt form of theendothelin receptor antagonist,(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid.

BACKGROUND OF THE INVENTION

The compound,(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid is included within the large generic class of compounds disclosedand claimed in the co-pending PCT application PCT/US 9612581. Thecompounds are described as existing in either the free acid form or as“pharmaceutically acceptable salts”. Example 2 of PCT/US 9612581 givesthe method of producing the diacid,(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid. The activity of the compound as an endothelin receptor antagonistis described in PCT/US 9612581.

SUMMARY OF THE INVENTION

The invention comprises the monoargininyl salt of(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid) which surprisingly has been found to have increasedbioavailability as compared to the disodium salt ordissolution-enhancing formulations of the diacid i.e., where thesurfactant Tween 80 has been incorporated either alone and incombination with the buffering agent N-methylglucamine.

The invention further constitutes pharmaceutical compositions of themonargininyl salt of(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid) and its use as an endothelin receptor antagonist which is usefulin the prevention or treatment of a variety of cardiovascular and renaldiseases including but not limited to: hypertension, acute and chronicrenal failure, cyclosporine induced nephrotoxicity, benign prostatichypertrophy, pulmonary hypertension, migraine, stroke, subarachnoidhemorrhage, cerebrovascular vasospasm, myocardial ischemia, angina,congestive heart failure, unstable angina, coronary vasospasm andmyocardial salvage, the sequelae of diabetes including but not limitedto: atherosclerosis, diabetic nephropathy, diabetic retinopathy,retinopathy, diabetic macrovascular disease; and as an adjunct inangioplasty for prevention of restenosis.

This invention further constitutes a method for antagonizing endothelinreceptors in an animal, including humans, which comprises administeringto an animal in need thereof an effective amount of a compound ofFormula (I).

DETAILED DESCRIPTION OF THE INVENTION

The compound of this invention is represented by structural Formula (I):

The aqueous solubility of the diacid of the compound of Formula (I) isvery limited under acidic conditions. The solubility is less than 0.1ug/mL at pH<5.34. For compounds possessing a pH-dependent solubilityprofile of this type, it is not uncommon to find a dissolution-ratelimiting component to the oral bioavailability. For the diacid ofFormula (I), this contention is supported by the finding that the oralbioavailability in dogs, following the intraduodenal dosing of anon-aqueous solution of a compound of Formula (I) diacid, wasapproximately 15% which is close to the theoretical maximum value of 18%(the enterohepatic extraction ratio is 0.82). In contrast, the oralbioavailability in dogs, following the oral dosing of a simple capsuleformulation of the diacid of Formula (I), was only 3.9%. In an attemptto overcome any dissolution-rate limitation and hence enhance the oralbioavailability, alternate salt forms were studied.

It has now unexpectedly been found that the newly prepared monoargininylsalt of Formula (I) has increased bioavailability as compared to thedisodium salt or dissolution-enhancing formulations of the diacid i.e.,where the surfactant Tween 80 has been incorporated either alone and incombination with the buffering agent N-methylglucamine. The mean percentoral bioavailability was 13.3% which is comparable to the intraduodenaldosing of a non-aqueous solution of the disodium form. Comparative datafor the bioavailability studies is found in Table 1.

TABLE 1 Bioavailability Studies Conducted in Dogs Using FormulationApproaches and SaIt Formd of Formula (I) Form Formulation Mean % OralDosed Dosed Bioavailability ± SD Disodium Salt Non-Aqueous Solution^(a)14.5^(b) Diacid Capsule^(c) 3.9 ± 0.5 Diacid Capsule with 0.5% Tween80^(d) 2.3 ± 3.7 Diacid Capsule with 0.5% Tween 80 & 6.3 ± 5.3 2 MEqNMG^(d) Disodium Salt Capsule^(c) 6.2 ± 4. 1 Monoargininyl Capsule^(c)13.8 ± 7.6  Salt ^(a)Solution contained 99% PEG 400 and 1% DMSO and wasdosed intraduodenally. ^(b)Dosed in only 2 animals. ^(c)Formulationsalso contain microcrystalline cellulose as a diluent. ^(d)Formulationsalso contained some microcrystalline cellulose, lactose, and starch.

As shown in Table 2, the monoarginine salt was minimally hygroscopicabsorbing only 2.3% moisture over a relative humidity range of 0 to 90%.It was slightly more hygroscopic than the diacid form and considerablyless hygroscopic than the amorphous disodium salt form. Acceleratedstability studies, performed on the monoarginine salt, show that it ischemically stable for at least three weeks when stored at 50° C.

TABLE 2 Hygroscopicity Studies for the Diacid and the Disodium andMonoargininyl Salt Forms of Formula (I) % Relative % Moisture Uptake at250Ca Humidity Diacid Disodium Monoarginine 0 0 0 0 10 0.077 1.849 0.26120 0.088 2.838 0.382 30 0.126 3.821 0.526 40 0.184 5.013 0.689 50 0.2456.773 0.827 60 0.316 10.188 0.982 70 0.395 15.919 1.092 80 0.509 22.4721.434 90 0.518 33.671 2.334 ^(a) Measured on a dynamic vapor sorptionanalyzer as percentage weight gain in the sample.

Pharmaceutical compositions having endothelin receptor antagonistactivity which comprises a pharmaceutical carrier containing and activebut non-toxic quantity of the monoargininyl salt of a compound ofFormula (I) are also objects of this invention.

In order to use the monoargininyl salt of a compound of the Formula (I)for the treatment of humans and other mammals it is normally formulatedin accordance with standard pharmaceutical practice as a pharmaceuticalcomposition.

The monoargininyl salt of Formula (I) may be administered in a standardmanner for the treatment of the indicated diseases, for example orally,parenterally, sub-lingually, transdermally, rectally, via inhalation,via ocular administration, or via buccal administration.

The monoargininyl salt of Formula (I) when given orally can beformulated as a syrup, tablet, capsule and lozenge. A syrup formulationwill generally consist of a suspension or solution of the compound in aliquid carrier for example, ethanol, peanut oil, olive oil, glycerine orwater with a flavoring or coloring agent. Where the composition is inthe form of a tablet, any pharmaceutical carrier routinely used forpreparing solid formulations may be used. Examples of such carriersinclude magnesium stearate, terra alba, talc, gelatin, agar, pectin,acacia, cellulose, mannitol, stearic acid, starch, lactose and sucrose.Where the composition is in the form of a capsule, any routineencapsulation is suitable, for example using the aforementioned carriersin a hard gelatin capsule shell. Where the composition is in the form ofa soft gelatin shell capsule any pharmaceutical carrier routinely usedfor preparing dispersions or suspensions may be considered, for exampleaqueous gums, celluloses, silicates or oils and are incorporated in asoft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension ofthe compound in a sterile aqueous or non-aqueous carrier optionallycontaining a parenterally acceptable oil, for example polyethyleneglycol, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that may be administered as a dry powder or inthe form of an aerosol using a conventional propellant such asdichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises the monoargininyl salt ofFormula (I), with a binding and/or lubricating agent, for examplepolymeric glycols, gelatins, cocoa-butter or other low melting vegetablewaxes or fats or their synthetic analogues.

Typical transdermal formulations comprise a conventional aqueous ornon-aqueous vehicle, for example a cream, ointment, lotion or paste orare in the form of a medicated plaster, patch or membrane.

Typical ophthalmic formulations are isotonic solutions buffered toneutral pH in the range of 6.5 to 7.8, for example a citrate buffer madeisotonic with sodium chloride.

Preferably the composition is in unit dosage form, for example a tablet,capsule or metered aerosol dose, so that the patient may administer tothemselves a single dose.

Each dosage unit for oral administration contains suitably from 0.1 mgto 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosageunit for parenteral administration contains suitably from 0.1 mg to 100mg, of the monoargininyl salt of Formula (I) or a pharmaceuticallyacceptable salt thereof calculated as the free acid. Each dosage unitfor intranasal administration contains suitably 1-400 mg and preferably10 to 200 mg per person. A topical formulation contains suitably 0.01 to1.0% of the monoargininyl salt of a compound of Formula (I).

The daily dosage regimen for oral administration is suitably about 0.01mg/Kg to 40 mg/Kg, of the monoargininyl salt of Formula (I) or apharmaceutically acceptable salt thereof calculated as the free acid.The daily dosage regimen for parenteral administration is suitably about0.001 mg/Kg to 40 mg/Kg, of the monoargininyl salt of the Formula (I) ora pharmaceutically acceptable salt thereof calculated as the free acid.The daily dosage regimen for intranasal administration and oralinhalation is suitably about 10 to about 500 mg/person. The activeingredient may he administered from 1 to 6 times a day, sufficient toexhibit the desired activity.

No unacceptable toxicological effects are expected when the compound ofthe invention is administered in accordance with the present invention.

The biological activity of the monoargininyl salt of Formula (I) isdemonstrated by the following test:

Bioavailability Study

Four male beagle dogs (approximate weight; 10-15 kg) were used. Thestudy was conducted as a crossover design on three separate study days,and the animals were allowed to recover for one week between each leg ofthe experiment. A CBC screen was performed on the animals before eachstudy day to obtain baseline values and assure hematological recovery.On each study day, a catheter was placed in a cephalic vein for bloodsampling. On study day three a catheter was also placed in a saphenousvein for i.v. administration. The animals were fasted overnight prior totreatment, and food was prepared after taking the 240 min blood samples.On study days one and two (oral dosing), the dogs were restrained inslings for approximately 1 hour and then transferred to metabolismcages. On study day three, the dogs were restrained in slings forapproximately 2 hours during the study and then transferred tometabolism cages. The animals were housed in individual cages inunidirectional air flow rooms with controlled temperature (22±2° C.) andrelative humidity (50±10%) and 12 h light/dark cycles (0600-1800). Thedogs were acclimatized for at least 5 days prior to the experiment andprovided food, except for the overnight period prior to dosing. Filteredtap water was supplied ad libitum.

The animals were fasted overnight prior to administration of thecompound. Food was provided after drawing the 240 min blood samples.

Preparation of Dose Solutions and Dosing Procedures

For the first oral administration, solid monoarginyl salt of Formula (I)was triturated by glass mortar and pestle to obtain a uniform particlesize. The compound and Avicel PH 102 was mixed as a 50:50 ratio andpacked into a white opaque gelatin capsule (size 00).

For the second oral administration, solid di-acid of Formula (I) wastriturated by lass mortar and pestle to obtain a uniform particle size.The compound and Avicel PH 102 was mixed as a 50:50 ratio and packedinto a white opaque gelatin capsule (size 00).

The dose solution for i.v. administration will be prepared in injectablesaline Solution containing less than 3% ethanol.

On study day one, two animals received the mono-argininyl salt ofFormula (I)(16.2 umol [10 mg]/kg target dose) in a gelatin capsule, andthe other two animals received the di-acid of Formula (I)(16.2 umol [10mg]kg target dose) in a gelatin capsule.

On study day two, the same formulations were used, but the study animalswere crossed over to receive the opposite formulation that they weregiven on day 1.

On study day three, the dogs received a compound of Formula (I) (0.5mg/mL in injectable saline) as a 90 min intravenous infusion (2.43 umol[1.5 mg]/kg target dose, 2 mL/kg/h, 3 mL/kg total volume).

Blood Sample Collection

Blood samples (approximately 0.25 mL) were collected into 1 mL syringesfrom the cephalic vein catheter at the approximate times and volumesshown in the tables below and transferred into heparinized 1.5 mLEppendorf tubes. Plasma (50 uL) was isolated from the blood samples bycentrifugation, transferred to new Eppendorf tubes and quick frozen ondry ice. Extra plasma was also frozen and saved. All samples were storedat or about −70° C. until analyzed.

Approximate Sample Times Following Oral Administration Time 0 5 15 30 4560 90 120 180 240 360 480 600 720 1440 (min) Total blood withdrawn:Approximately 11.5 mL (includes pre-draw) Approximate Sample TimesFollowing Initiation of Intravenous Infusion Time 0 15 30 45 60 75 90 9295 105 120 180 240 360 480 600 720 1440 (min) ↑ ↑ Start infusion Endinfusion Total blood withdrawn: Approximately 13.5 mL (includespre-draw)

Concentrations of the compound of Formula (I) in plasma samples werequantified using an HPLC/MS/MS method.

EXAMPLE 1

Preparationof(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicAcid, Monoargininyl Salt

L-Arginine (152.25 g, 0.87 mole) was added, with stirring, to a mixtureof methanol (4 L) and water (40 mL) which had been heated to about 40°C. The temperature was then raised to 50° C. and the suspension wasstirred for 15 minutes. SB 247083 (525 g, 0.85 mole) was then added, andthe suspension was heated to about 55° C. After all of the soliddissolved, the solution was filtered. The filtrate was stirred andslowly cooled to room temperature. Upon reaching room temperature, thefiltrate was stirred for about 1 hour. The resulting crystals werefiltered off then washed with about 200 ml of methanol. Then thecrystals were air dried for about 30 minutes then vacuum dried at 50 to60° C.

Sharp endotherm onsets at 214.4 C (delta H=85 J/g) from DSC.

EXAMPLE 2

Preparation of a pharmaceutical composition of(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2.3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicAcid Monoargininyl Salt

Formulations for pharmaceutical use incorporating the compound of thepresent invention can be prepared in various forms and with numerousexcipients. Examples of such formulations are given below.

Inhalant Formulation

A compound of Formula (I), (1 mg to 100 mg) is aerosolized from ametered dose inhaler to deliver the desired amount of drug per use.

Ingredients Amount per Tablet 1. Active ingredient 40.0 mg (Cpd of Form.I) 2. Lactose 40.0 mg Microcrystalline Cellulose 16.0 mg 4. SodiumStarch Glycolate 4.0 mg 5. Povidone 5.3 mg 6. Microcrystalline Cellulose89.2 mg 7. Sodium Starch Glycolate 4.0 mg 8. Mg Stearate 1.5 mg Total200.0 mg

Procedure for Tablets:

Step 1 Blend ingredients No. 1, 2, 3, 4, and 5 in a suitablemixer/blender.

Step 2 While mixing, add water to the blend to produce a wet mass thencontinue to granulate until a suitable wet granulation is obtained.

Step 3 Pass the wet mass through a mill fitted with an appropriatescreen.

Step 4 Dry the granules in a fluid-bed dryer at an appropriate inlettemperature.

Step 5 Screen the granulate through a mill fitted with an appropriatescreen.

Step 6 Blend the granules with ingredients No. 6, 7, and 8.

Step 7 Compress the formulation on an appropriate tablet press.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of Formula (I) inpolyethylene glycol with heating. This solution is then diluted withwater for injections Ph Eur. (to 100 ml). The solution is then steriledby filtration through a 0.22 micron membrane filter and sealed insterile containers.

What is claimed is: 1.(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoic acid, monoargininyl salt.
 2. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 3. A composition according to claim 2 for oraladministration.
 4. A compound of claim 1 for use as an endothelinreceptor antagonist.
 5. A method of treatment of diseases caused by anexcess of endothelin comprising administering to a subject in needthereof, an effective amount of an endothelin receptor antagonist ofclaim
 1. 6. A method of treating hypertension, renal failure orcerebrovascular disease which comprises administering to a subject inneed thereof, an effective amount of a compound of claim
 1. 7. A methodfor the treatment of chronic renal failure which comprises administeringto a subject in need thereof, an effective amount of a compound of claim1.
 8. A method of treatment of benign prostatic hypertrophy whichcomprises administering to a subject in need thereof, an effectiveamount of a compound of claim
 1. 9. A method of treatment of congestiveheart failure which comprises administering to a subject in needthereof, an effective amount of a compound of claim
 1. 10. A method oftreatment of unstable angina, coronary vasospasm and myocardial salvage,which comprises administering to a subject in need thereof an effectiveamount of a compound of claim
 1. 11. A method of preventing or treatingrestenosis which comprises administering to a subject in need thereof,an effective amount of a compound of claim
 1. 12. A method of treatmentof pulmonary hypertension which comprises administering to a subject inneed thereof, an effective amount of a compound of claim
 1. 13. A methodof treatment of atherosclerosis which comprises administering to asubject in need thereof, an effective amount of a compound of claim 1.14. A method of preventing and treating the sequelae of diabetes whichcomprises administering to a subject in need thereof, an effectiveamount of a compound of claim
 1. 15. A method of treatment of stroke orsubarachnoid hemorrhage which comprises administering to a subject inneed thereof, an effective amount of a compound of claim
 1. 16. Aprocess for preparing a compound of claim 1 by adding L-Arginine to(E)-3-[1-n-Butyl-5-[2-(2-carboxyphenyl)methoxy-4-chlorophenyl]-1H-pyrazol-4-yl]-2-[(5-methoxy-2,3-dihydrobenzofuran-6-yl)methyl]-prop-2-enoicacid.